ABSTRACT
Affinity chromatography on immobilized Fe(3+) ions--immobilized-metal-ion affinity chromatography (IMAC) method--was used for the determination of pepsin and pepsinogen phosphorylation. IMAC is a very powerful method for detailed studies of proteins. Dephosphorylation of the pepsinogens and pepsins has no effect on their proteolytic ability. For this reason, the determination of proteolytic activity was used for the detection of pepsinogen (pepsin) presence in the collected fractions as a very suitable and specific method. Pepsins and their zymogens probably have the same amounts of phosphate ions in their molecule. The exact definition of conditions is very important for the prepurification of the proteinases and for their analysis.
Subject(s)
Gastric Mucosa/enzymology , Pepsinogen A/metabolism , Amino Acid Sequence , Buffers , Chromatography, Affinity/methods , Chromatography, Gel/methods , Chromatography, Ion Exchange/methods , Ferric Compounds , Humans , Indicators and Reagents , Ligands , Metals , Oligopeptides , Pepsin A/metabolism , PhosphorylationABSTRACT
A new combination of chromatographic and electrophoretic methods has been developed for better separation and characterization of human pepsinogens. Pepsinogens isolated from the gastric mucosa of patients with gastric cancer have been separated using fast-protein liquid chromatography (FPLC) on an ionex Uno-Q1 column. Proteolytic active fractions were firstly immunodetected by monoclonal antibodies against PGA and PGC using ELISA and then separated by isoelectric focusation in the acidic pH 2.5-5 gradient with an excellent resolution. The combination FPLC and ELISA followed by IEF enabled to separate ten pepsinogen isoforms. This technique is very suitable for studies of the pepsinogen polymorphism and its role in the gastric diseases.
Subject(s)
Chromatography, Liquid/methods , Electrophoresis, Polyacrylamide Gel/methods , Pepsinogens/genetics , Polymorphism, Genetic , Enzyme-Linked Immunosorbent Assay , Humans , Isoelectric Focusing , Pepsinogens/metabolism , Stomach Neoplasms/metabolismABSTRACT
Electrotitration curves (ETC) of a marker protein mixture, pH 2.5-5.65, and human pepsinogens were performed in an agarose gel, containing 2% acid carrier ampholytes, forming a pH range of 2.5-5. Although the establishment of the pH gradient by isoelectric focusing was not quite complete and linear, both biochemically and immunochemically different types of pepsinogen C (PGC) and pepsinogen A (PGA) zymogens as well as the acid isoelectric points (pI) marker proteins were separated with good resolution. Three main fractions of PGA (Pg3, Pg4, and Pg5) were detected. To obtain an exact determination of the pepsinogen pIs, a simple and very fast 10 s pressure blot technique was applied. Human pepsinogens were separated alone or mixed with pI marker proteins in the pH range 2.4-5.65. No effect of the markers was observed on the pepsinogen migration. To visualize the different protein samples in the gel and on nitrocellulose membrane, we have used colloidal gold (AuroDye) staining, proteolytic activity, and immunostaining with monoclonal antibodies anti PGA and PGC. The described method shows an ability to separate proteins at acidic conditions with a resolution comparable to isoelectric focusing with immobilized pH gradients, but much faster, easier, and cheaper. In addition, the technique allows us to determine precise and exact pI values, and is suitable for studies of the pepsinogen polymorphism and its role in gastric diseases.
Subject(s)
Electrophoresis/methods , Pepsinogens/analysis , Humans , Hydrogen-Ion Concentration , Stomach/chemistryABSTRACT
Phosphorylation is one of the most important ways of posttranslational modification of proteins that enables regulation of many cell physiological processes (transport, proliferation, differentiation). This reversible and very fast process is involved in all phases of cell division: in transition from G1 to S phase, progression of cells during S phase and entry into M phase. The physiological function of oncoproteins and tumor suppressor proteins that are involved in gene expression and replication are also regulated by phosphorylation. Many growth factors and their receptors are encoded by oncogenes which are mutated or overexpressed in a variety of human tumors. Mutation or overexpression of these oncogenes leads to uncontrollable cell division and transformation of normal cells to malignant. Cancer can be considered as a disease of the signalling pathways.
Subject(s)
Neoplasms/physiopathology , Phosphoprotein Phosphatases/physiology , Protein Kinases/physiology , Proteins/physiology , Animals , Cell Cycle/physiology , Humans , PhosphorylationABSTRACT
The human gastric mucosa contains two main groups of aspartic proteinases, pepsin (EC 3.4.23.1) and gastricsin (EC 3.4.23.3), which differ by their structural, kinetic and immunological characteristics. The ratios between human aspartic proteases are important from the diagnostic point of view. Rabbit polyclonal antisera against human pepsinogen A and pepsinogen C (progastricsin) were obtained and tested for clinical purposes. Immunoblotting procedure seems to be a simple and sufficiently sensitive method for qualitative determination of pepsinogens in human gastric mucosa.
